Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death globally. Tumors driven by expression of oncogenic Kras account for approximately 25% of NSCLC subtypes and, for these tumors in particular, effective chemotherapies are lacking. One possible explanation for the failure of standard chemotherapies in these tumors is the cellular heterogeneity that exisists within tumors. The goal of this proposal is to understand the cellular heterogeneity in Kras-driven lung adenocarcinoma. Specifically, I will explore the role of the Wnt and R-spondin/Lgr5 family signaling pathways as a paracrine regulators of cellular de-differentiation and stemness in the tumors. To do so, I will employ a series of novel tools in a sophisticated mouse model of Kras- driven lung adenocarcinoma as well as in human lung adenocarcinoma cell lines. I propose to test the role of the Wnt and R-spondin/Lgr pathways in lung tumor initiation and progression, as well as potential targets of therapy. To do this, I will use small molecule inhibitors of Wnt synthesis or dual-promoter lentiviruses to silence key components of the pathway, including Lgr5 family receptors, using RNAi. Furthermore, novel Wnt reporter lentiviruses or knock-in alleles expressing Lgr5 or Lgr6 driven and tamoxifen-activatable CreER recombinase will be used to perform lineage-tracing experiments that allow me to track the fate of or ablate the putative lung adenocarcinoma stem cells in a tumor model that undergoes natural tumor progression and is not based on cell line transplantation, a caveat in most of the current literature. Use of Wnt-responsive and Lgr5/6 reporters will also enable me to isolate the putative lung adenocarcinoma stem cells for gene expression profiling, which may lead to the discovery of novel stem cell markers and druggable pathways. Elucidating the molecular mechanisms that regulate cell (differentiation) states in cancer will provide novel markers for mapping the cellula landscape of tumors; some will prove to be useful targets for pharmacological intervention, which will eventually improve treatment outcomes in this largely intractable disease. Thus, I feel that this proposal is fully aligned with the mission of the National Cancer Institute. In this application I also propose an extensive training program that is designed to facilitate my transition to an independent Principal Investigator position. The research environment in the Jacks Laboratory, MIT, and the surrounding area offers unparalleled opportunities for scientific discussion, collaboration and training. I currently supervise an undergraduate student and a technical assistant that work directly with me on experiments pertaining to my research. This is an incredible experience that will endow me with many of the necessary skills to manage an independent laboratory. The scientific community at MIT, the Broad Institute, and Harvard Medical School offers countless courses, seminars and workshops that will continue to foster my scientific development. I have been fortunate in having been able to assemble a dream team of Mentors (Dr. Jacks and Dr. Weinberg) and Consultants (Dr. Clevers and Dr. Scadden), who will provide me with the necessary guidance and support throughout the entire duration of the K99/R00 Award. Importantly, my Primary Mentor Dr. Jacks will allow me to take all of my current and proposed research with me to serve as the foundation of my future research program. The research proposed within this application has been shaped by my experiences in studying cell-cell interactions controlling (tumor) angiogenesis and lymphangiogenesis, as well as by the past 2 years in the Jacks Laboratory learning novel methodology and concepts. I intend to start an independent research program that will capitalize on these powerful in vivo systems. I have already demonstrated my independence by creating a project in a field not previously studied in our lab as well as by obtaining independent funding in the form of Fellowships and small project grants. This, in combination with the large number of reagents that I have developed, provides me with the momentum needed to complete the proposed program. For the long-term, I am confident that these experiments will provide a solid foundation on which my research program can be built upon. I look forward to mentoring students and postdocs that share my passion for cancer research.